The long-term goal of our research is to construct and characterize a realistic three-dimensional model of the brain extracellular space (ECS), in order to predict the impact of microstructural changes on the transport of signaling molecules, nutrients and therapeutic agents. ECS comprises the narrow channels that separate brain cells but cannot be directly visualized in the living brain. It is essential for normal brain function and influences many critical processes including intercellular signaling, nutrient delivery and neurotrophic effects. Significantly, the ECS also forms the final route for all drug delivery to brain cells. To develop quantitative understanding of any of these diffusion-mediated processes, essential structural parameters of the complex ECS environment must be identified and characterized. Traditional diffusion measurements, made over relatively large distances, extract two macroscopic parameters, volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by the ECS, and tortuosity quantifies average hindrance imposed on diffusing molecules by the complex ECS environment. The concentration of a diffusing substance is primarily influenced by volume fraction while tortuosity imposes delays in the timing. It has been taken for granted that these parameters remain constant over all diffusion distances. However, we have recently discovered that diffusion in the brain is transiently anomalous over distances of a few tens of micrometers. This means that, over this distance, the rate of diffusion depends on time and generally is faster than currently believed. To explore the phenomenon of a transiently anomalous diffusion, we introduce the concept of a Dynamic Microdomain (DM), defined as the largest volume of the brain tissue in which the anomalous diffusion is observed. The size of the DM will depend on the local structure and can change in response to various stimuli. In Aim 1, we propose to develop a Fast Optical Tracking of Diffusion (FOTOD) method to measure DM size and analyze diffusion within it. FOTOD will be equally applicable when normal diffusion occurs in a dynamically changing ECS, e.g., during spreading depression (Aim 1) or synchronous neuronal activity (Aim 4). Aims 2-4 will explore several physiologically important aspects of the DM structure with this new methodology. Aim 2 determines that structural plasticity of the astrocytic processes induced by beta2- adrenergic neuron-glia signaling represents an, as yet unrecognized, mechanism that modulates cellular communication in the visual cortex. The astrocytic processes act by altering the DM diffusion properties. Aim 3 shows how negatively-charged perineuronal matrix nets attract polyvalent cations (e.g., calcium) but repulse polyvalent anions, thereby acting as charge discriminators within the DMs. In Aim 4, scaling theory applied to the diffusion of flexible polymers estimates the average width of ECS pores. Very few estimates of this basic parameter exist in living brain, yet the characteristic pore width is essential for the development of drug carriers, and for any realistic model of DMs. PUBLIC HEALTH RELEVANCE: Brain cells, comprising neurons and glia, are surrounded by extracellular space (ECS), a system of interconnected pores that channels chemical signals between cells and is an essential route for delivery of nutrients and drugs. This project combines experiments to measure diffusion in brain tissue with mathematical modeling to characterize the microstructure of the ECS and how it is regulated. The results will be important both for understanding how altered ECS structure in neuropathological states disrupts the chemical traffic of the brain, and for designing effective strategies to deliver drugs in patients suffering from neurological disorders and brain tumors.